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Gadolinium chloride decreases circulating monocytes and neutrophils following ischemia and reperfusion. Rats were treated with either saline or GdCl3 (20 μmol/kg) 15 minutes prior to a Sham procedure or a 30 minute period of regional ischemia and 2 hours reperfusion. A) Increase in monocytes. B) Increase in neutrophils. Data mean ± SD, n = 3-6/gp, + = p < 0.05, Sham control vs. Ischemia-Reperfusion (IR) control,* = p < 0.05, IR Control vs. IR GdCl3, ± = p < 0.05, Sham Control vs. Sham GdCl3, § = p < 0.05, IR GdCl3l vs. Sham GdCl3
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The lanthanide cation, gadolinium (GdCl3) protects the myocardium against infarction following ischemia and reperfusion. Neutrophils and macrophages are the main leukocytes responsible for infarct expansion after reperfusion. GdCl3 interferes with macrophage and neutrophil function in the liver by decreasing macrophage secretion of inflammatory cyt...
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Aim
Myocardial infarction (MI) remains a major cause of death and disability worldwide, despite available reperfusion therapies. Inflammatory signaling is considered nodal in defining final infarct size. Activation of the innate immune receptor toll-like receptors (TLR) 9 prior to ischemia and reperfusion (I/R) reduces infarct size, but the consequ...
Activation of heme oxygenase-1 (HO-1) has been proved to reduce damages to the liver in ischemia reperfusion injury. The objective of present study was to determine whether clinic relevant doses of isoflurane treatment could be sufficient to activate HO-1 inducing, which confers protective effect against hepatic ischemia-reperfusion injury.
The hep...
To evaluate the effects of hyperbaric oxygen (HO) therapy in the protection against liver ischemia/reperfusion injury.
Thirty-two male Wistar rats were divided into four groups of eight animals each: group A - laparotomy and liver manipulation, group B - liver ischemia and reperfusion, group C - HO pretreatment for 60 min followed by liver ischemia...
Background
Nowadays, treatments for cholestasis remain largely nonspecific and often ineffective. Recent studies showed that inflammatory injuries and oxidative stress occur in the liver with cholestasis. In this study, we would use corilagin to treat the animal model of acute cholestasis in order to define the activity to interfere with inflammati...
Although recent preclinical and clinical studies have demonstrated that recombinant human relaxin (rhRLX) may have important therapeutic potential in acute heart failure and chronic kidney diseases, the effects of acute rhRLX administration against renal ischaemia/reperfusion (I/R) injury have never been investigated. Using a rat model of 1-hr bila...
Citations
... Ingestion of gadolinium by phagocytes induces chelation of the gadolinium and phosphate complex, leading to selective depletion and/or inhibition of its antipathogenic and immune-regulatory functions [46][47][48]. Previous studies have found that GdCl3 can influence the Kupffer cell-related pathophysiological processes of drug-induced liver toxicity, lung inflammation, and ischemia-reperfusion [49][50][51][52][53]. In a CLP-induced severe infection rat model, it was found that the inflammatory fac-tors levels in the serum and liver were decreased when rats were pretreated with GdCl3 [42], while Ravinder et al. found that the serum cytokine and chemokine levels in the GdCl3-pretreated CLP mice group were not different from the CLP mice group without GdCl3-pretreatment [20]. ...
Background. Liver macrophages play an important regulatory role in the inflammatory response of liver injury after severe infection. Interleukin- (IL-) 27 is an inflammatory cytokine that plays an important role in diseases caused by bacterial infection. However, the relationship between IL-27 and liver macrophages in liver injury after severe infection is not yet clear. Methods. A cecal ligation puncture (CLP) model was established in wild-type (WT) and IL-27 receptor- (WSX-1-) deficient (IL-27r-/-) mice, and recombinant IL-27 and gadolinium chloride (GdCl3) were injected into WT mice in the designated groups. The serum and liver IL-27, IL-6, tumor necrosis factor alpha (TNF-α), and IL-1β expression levels were evaluated by ELISA, quantitative PCR, or Western blotting; serum ALT and AST were detected by detection kits; and the severity of liver damage was evaluated by hematoxylin and eosin staining and the TUNEL assay of the liver tissue from the different groups. Liver macrophage polarization was evaluated by immunofluorescence. In addition, the polarization of peritoneal macrophage was evaluated by flow cytometry. Results. The serum and liver IL-27 expression levels were elevated in WT mice after CLP-induced severe infection, which were consistent with the changes in HE scores in the liver tissue. The levels of serum ALT, AST, liver IL-6, TNF-α, and IL-1β mRNA and liver pathological injury scores were further increased when pretreated with recombinant IL-27 in WT mice, but these levels were decreased in IL-27r−/− mice after CLP-induced severe infection compared to WT mice. In WT mice pretreated with GdCl3, liver pathological scores, serum ALT and AST, TUNEL-positive cell proportion from liver tissues, liver IL-27 expression, and the liver macrophages M1 polarization proportion decreased after CLP; however, the serum IL-27, IL-6, TNF-α, and IL-1β levels and the pathological lung and kidney scores were not significantly changed. When supplemented with exogenous IL-27, the liver pathological scores, serum ALT, AST, TUNEL-positive cell proportion of liver tissues, liver IL-27 expression, and the liver macrophage M1 polarization proportion increased. The in vitro, IL-27 expression increased in peritoneal macrophages when stimulated with LPS. Recombinant IL-27 together with LPS promoted the elevations in IL-6, TNF-α, and IL-1β levels in supernatant and the M1 polarization of peritoneal macrophages. Conclusion. IL-27 is an important cytokine in the inflammatory response to liver injury after severe infection. The reduction of liver injury by gadolinium chloride in severe infection mice models may relate to the inhibition of liver IL-27 production. These changes may be mainly related to the decrease of liver macrophages M1 polarization. IL-27 may have a positive feedback on these macrophages.
1. Introduction
Acute liver dysfunction is a concomitant manifestation of many critical illnesses.
Liver dysfunction is sometimes closely related to poor prognosis in critical patients [1, 2]. Severe infection is one of the most important causes of liver dysfunction in critical patients [3]. In severe infection, the liver plays crucial roles in pathogen defense, clearance, and mediating inflammatory responses in the pathophysiology process [4]. However, this involvement is a double-edged sword: the liver removes bacteria and toxins and can also cause inflammation, immune suppression, and organ damage due to an overwhelming systemic inflammatory storm [5]. Interleukin- (IL-) 27 is a heterodimeric cytokine of the IL-12 family composed of the Epstein-Barr virus- (EBV-) induced gene 3 (EBI3) and the p28 subunit and plays an important regulatory role in the inflammatory responses of infectious disease [6, 7]. IL-27 is a biphasic regulator, acting as a pro- or anti-inflammatory factor under different conditions. It is involved in the induction of IL-10 production, Foxp32, and T-regulatory 1 (Tr1) cells and plays an anti-inflammatory effect [8, 9]; in contrast, it can act as a promotor in peritonitis and in ConA-induced liver injury animal models [10, 11].
Liver macrophages, especially Kupffer cells (KCs), play an important role in the regulation of inflammatory after severe infection [12]. In severe infection, activated Kupffer cells can release large amounts of cytokines, chemokines, prostaglandins, leukotrienes, and complement factors, playing an important role in regulating liver and systemic inflammation and promoting liver damage [13–15]. As IL-27 is mainly produced by macrophages and DCs [16], we speculate that macrophages may regulate liver damage by producing IL-27 in severe infections.
Gadolinium chloride (GdCl3) is an inhibitor of Kupffer cells [17]. It has been reported that GdCl3 contributes to the reduction of liver damage by inhibiting the activation of KCs according to many types of liver injury, including ethanol, dimethylnitrosamine, carbon tetrachloride administration (CCl4), and cadmium-induced liver injury [18, 19]. GdCl3 treatment reduced the proinflammatory cytokine levels of liver, including IL-6, TNF-α, IL-1β, and alleviated liver injury after CLP [20]. However, the exact mechanisms involved in the reduction of liver injury after GdCl3 treatment are not yet clearly known. Therefore, the aim of this study is to investigate whether GdCl3 can reduce liver injury by affecting IL-27 production.
2. Materials and Methods
2.1. Animal Models
Male C57BL/6 J mice aged 8–10 weeks were used to establish the cecal ligation puncture (CLP) model [21]. The sham group mice have underwent exactly the same procedures as the CLP group, without the cecum ligation and puncture. Wild-type (WT) C57BL/6 J mice were obtained from the Experimental Animal Center of Chongqing Medical University, and IL-27r-/- (WSX-1 knockout) C57BL/6 J mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All the mice were raised under the specific pathogen-free conditions. The animal experiments were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University.
2.2. GdCl3 and Recombinant IL-27 Treatment
In the animal experiments, mice were intravenously injected (via tail vein) with GdCl3 (20 mg/kg body weight, Sigma-Aldrich) 24 h before CLP and then injected intraperitoneally with recombinant mouse IL-27 [7] (1 μg; ProSpec-Tany TechnoGene Ltd, Ness-Ziona, Israel) 2 h before CLP; all the groups were observed for 24 h after CLP injection. In the cell experiments, peritoneal macrophages were treated with recombinant IL-27 (50 ng/mL) 2 h before the addition of lipopolysaccharide (LPS, 100 ng/mL; Sigma–Aldrich, St. Louis, MO, USA), followed by incubation for 12 h.
2.3. Cell Extraction and Culture
Intraperitoneal injection of 3% thioglycollate is being used in C57BL/6 J mouse for 3 days, then the peritoneal cavity of each mouse was lavaged with 20 mL of PBS. After that, three to five milliliters of abdominal cavity liquid was extracted with a syringe and injected into a sterile centrifuge tube, which was then centrifuged at 1000 r/min for 5 min. The collected macrophages were resuspended in the medium (RPMI-1640 with 10% fetal bovine serum, 100 U/mL penicillin, and 10 μg/mL streptomycin) and then incubated in a 24-well plate at the concentration of cells/ml with the same medium, in the incubator at 37°C in a humidified atmosphere containing 5% CO2. After 2 h of incubation, warm PBS was used to wash the plate, nonadherent cells were removed with PBS, and then the adherent cells were incubated in the same medium and cultivation environment as before. The total liver macrophages of mice in the WT-CLP group and WT-CLP with GdCl3 pretreatment group were extracted as described [22].
2.4. HE and TUNEL
The liver, lung, and kidney tissues of mice obtained from each group at the indicated time point were fixed in paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E) or using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit (apoptosis detection kit (Roche, Mannheim, Germany)) to evaluate the cell apoptosis. Every tissue section was analyzed by light microscopy under ×200 and ×400 magnifications, and dark brown stained cell is positive. The liver, lung, and kidney pathology scoring methods used to evaluate the degree of tissue damage were previously described [23–25]. The degree of apoptosis was quantified based on the proportion of TUNEL-positive cells, counted under ×400 magnification of the microscope, 5 fields of view were taken for each tissue section, and 200 cells were counted in each field of view; the proportion of positive cells was calculated, and the final result of each tissue section is the average of the positive cell proportion of 5 fields of view.
2.5. Quantitative Polymerase Chain Reaction Assay
We followed the methods of Dr. Fan et al. 2019 [26]. Quantitative PCR (qPCR) for EBI3, P28, IL-6, TNF-α, and IL-1β was performed following the protocol by GeneCopoeia, Rockville, MD, USA. Total RNA was extracted from the liver tissue or cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Takara, Tokyo, Japan). The primer sequences were designed and chemically synthesized by Sangon Bio (Shanghai, China): EBI3 forward 5-GCT GCT CTT CCT GTC ACT TGC C-3 and reverse 5-TGA AGG ACG TGG ATC TGG TGG AG-3; p28 forward 5-CTG CTT CCT CGC TAC CAC ACT TC-3 and reverse 5-CTC TTC CTC CTT GTC CTC CTC CTC-3 (antisense); TNF-α forward 5-GCG ACG TGG AAC TGG CAG AAG-3 and reverse 5-GCC ACA AGC AGG AAT GAG AAG AGG-3; IL-6 forward 5-ACT TCC ATC CAG TTG CCT TCT TGG-3 and reverse 5-TTA AGC CTC CGA CTT GTG AAG TGG-3; IL-1β forward 5-TCG CAG CAG CAC ATC AAC AAG AG-3 and reverse 5-TGC TCA TGT CCT CAT CCT GGA AGG-3; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward 5-AGC GAG ACC CCA CTA ACA-3 and reverse 5-GGG GCT AAG CAG TTG GTG-3.
2.6. Western Blot Analysis
Following the methods of Dr. Fan et al. 2019 [26], protein samples were harvested from the liver tissue or cells by lysis buffer containing protease and phosphorylation inhibitors, and the protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). The protein samples were separated by 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked in QuickBlock™ Blocking Buffer for Western blotting (Beyotime Biotechnology) and then incubated with the following primary antibodies at 4°C overnight: rabbit polyclonal anti-IL-27-A antibody (p28) (1 : 500; Abcam, Cambridge, UK), rabbit polyclonal anti-IL-27-B (EBI3) antibody (1 : 500; AbKlean, Sangon Biotech, Shanghai, China), β-actin monoclonal antibody (1 : 1000), and β-tubulin monoclonal antibody (1 : 1000) (Boster Biological Technology, Wuhan, China); β-actin and β-tubulin were used as the sample loading controls. The next day, the membrane was incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit IgG (1 : 5000; ZSGB-BIO, Beijing, China) or goat anti-mouse IgG (1 : 1000; Boster Biological Technology, Wuhan, China) secondary antibody. Enhanced chemiluminescence was used to detect the proteins using a Chemiluminescent Detection Kit (Advansta, San Jose, CA, USA).
2.7. Cytokine and Liver Enzymes Assay
Following the methods of Dr. Fan et al. 2019 [26], IL-27 levels (R&D Systems, Minneapolis, MN, USA) in mouse serum and IL-6 (Boster Biological Technology, Wuhan, China) and TNF-α and IL-1β(R&D Systems) levels in cell supernatant were detected by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol.
Alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum were measured at 24 h after CLP and normal controls using detection kits (Nanjing JianCheng Bioengineering Institute, Jiangsu, China).
2.8. Immunofluorescence
The immunofluorescence method for detecting liver macrophages is as follows: first, frozen liver sections were recovered by PBS and then 0.3% Triton X-100 (50 μL) was applied for 15 minutes to rupture the cell membrane. Later, we used sodium citrate solution and heating for antigen retrieval and normal goat serum to block the section. After that, rabbit anti-mouse F4/80 polyclonal antibody was added to the section, and incubation was performed in a humid chamber at 4°C overnight. The next day, the section was kept for 1 h at room temperature, washed in PBS, and then the goat anti-rabbit PE-Cy3 secondary antibody was added for 1 h at room temperature. After that, the section was washed in TBST for 3 times and used sodium citrate solution and heating for antigen retrieval and normal goat serum to block the section again. And then, rabbit anti-mouse CD206 polyclonal antibody was added to the section, incubated in a humid chamber at 4°C overnight. The following day, goat anti-rabbit FITC secondary antibody was added, and the detailed steps were the same as before. After that, the section was washed in TBST for 3 times again and used sodium citrate solution and heating for antigen retrieval and normal goat serum to block the section one more time. Later, rabbit anti-mouse iNOS polyclonal antibody was added to the section, and incubation was performed in a humid chamber at 4°C overnight. The following day, the goat anti-rabbit PE-Cy5 secondary antibody was added, and the detailed steps were the same as before. After washed, the section was added with autofluorescence quencher for 5 minutes and washed with running water for 10 minutes. Then, 4,6-diamidino-2-phenylindole (DAPI) was used to dye the cell nuclei; after that, the section was washed for 3 times, and antifade mounting medium was used for sealing. Positive cells were counted and analyzed by fluorescence microscope under ×200 magnifications of the microscope, 5 fields of view were taken for each tissue section, the total macrophage numbers, the M1 type macrophages, and the M2 type macrophages numbers were counted separately, the proportion of M1 and M2 type macrophages were calculated, and the final result of each tissue section is the average of the M1 and M2 type macrophage proportion of 5 fields of view. The primary and secondary antibodies were purchased from Servicebio, Wuhan, China.
The immunofluorescence method has also been used to detect the expression of IL-27p28 in liver macrophages. The detailed steps were the same as before. Rabbit anti-mouse F4/80 polyclonal antibody and goat anti-rabbit CY3-TSA secondary antibody were used for identifying the macrophages. And rabbit polyclonal IL-27p28 antibody and goat anti-rabbit FITC secondary antibody were used for detecting the expression of IL-27p28. The expression of IL-27p28 in F4/80-positive cells was analyzed by confocal microscopy under ×200 magnifications of the microscope, and 5 fields of view were taken for each tissue section. Total F4/80-positive cells, both F4/80 and IL-27p28-positive cells, were counted in each view, and the proportion of IL-27p28-positive cells in total F4/80-positive cells wascalculated. The average result of the 5 fields of view is the final result of each tissue section.
2.9. Flow Cytometry
The membrane surface molecules of cells were stained with 3 μL/test of PE-conjugated anti-mouse CD86 MAb (Invitrogen, California, USA) or APC-conjugated anti-mouse CD206 MAb (Invitrogen, California, USA) for 30 min at room temperature in the dark as per the manufacturer’s instructions. The mean fluorescence intensity (MFI) of the cell surface molecules was assessed by flow cytometry (FCM).
2.10. Statistical Analysis
The data in this study are expressed as the (SDs), and the statistical significance of differences between two groups was statistically analyzed with the independent -test. The one-way ANOVA or two-way ANOVA analysis was used when three or more groups were being compared. All of the analysis were performed with SPSS 23.0 statistical software and GraphPad Prism 7.04. was considered to be statistically significant.
3. Results
3.1. IL-27 Expression Is Upregulated in Liver Damage Mice after CLP
To verify the relationship between IL-27 and liver injury in a CLP-induced severe infection mouse model, we first detected IL-27 levels in serum by ELISA and the expression levels of EBI3 and P28 (subunits of IL-27) in the liver tissue by q-PCR and Western blot. We found that, compared to the sham group, the serum level of IL-27 was increased after CLP (; Figure 1(a)), and the liver EBI3 and P28 mRNA (by q-PCR) and protein expression levels (by Western blot) were also upregulated after CLP (; Figures 1(b) and 1(c)). Meanwhile, the levels of the inflammatory factors IL-6, TNF-α, and IL-1β in the liver tissue, determined by q-PCR, were also increased at the same time point after CLP (; Figure 1(e)). The histological scores of liver injury and serum ALT and AST levels also increased under the same conditions (; Figures 1(d) and 1(f)).
(a)
... Considering that GdCl3 also affects neutrophils [21,22], the beneficial effects of GdCl3 treatment may also be influenced by the inhibition of cells. ...
... Strande et. al. [22] showed that GdCl3 treatment decreased circulating monocytes and neutrophils, and leukocyte infiltration into injured myocardium, and this seems be the case in the brain as well. Evidence suggests that GdCl3 interferes with macrophage and neutrophil function in the liver by decreasing macrophage secretion of inflammatory cytokines and toxic oxygen radicals [55] and by inhibiting neutrophil infiltration [56]. ...
Background:
In order to modulate microglial phenotypes in vivo, M1 microglia were depleted by administration of gadolinium chloride and the expression of M2 microglia was induced by IL-4 administration in an animal model of sepsis to better characterize the role of microglial phenotypes in sepsis-induced brain dysfunction. Methods: Wistar rats were submitted to sham or cecal ligation and perforation (CLP) and treated with IL-4 or GdCl3. Animals were submitted to behavioral tests 10 days after surgery. In a separated cohort of animals at 24 hours, 3 and 10 days after surgery, hippocampus was removed and cytokine levels, M1/M2 markers and CKIP-1 levels were determined.
Results:
Modulation of microglia by IL-4 and GdCl3 was associated with an improvement in long-term cognitive impairment. When treated with IL-4 and GdCl3, the reduction of pro-inflammatory cytokines was apparent in almost all analyzed time points. Additionally, CD11b and iNOS were increased after CLP at all time points, and both IL-4 and GdCl3 treatments were able to reverse this. There was a significant decrease in CD11b gene expression in the CLP+GdCl3 group. IL-4 treatment was able to decrease iNOS expression after sepsis. Furthermore, there was an increase of CKIP-1 in the hippocampus of GdCl3 and IL-4 treated animals 10 days after CLP induction.
Conclusions:
GdCl3 and IL-4 are able to manipulate microglial phenotype in an animal models of sepsis, by increasing the polarization towards an M2 phenotype IL-4 and GdCl3 treatment was associated with decreased brain inflammation and functional recovery.
... From the nuancing effects of alloying elements, the specific inhibition of Gd on cell recruitment has already reported in an in vivo ischaemia-reperfusion rat model [53] . The authors observed that GdCl 3 solution pre-treatment leads to a decrease in circulating immune cells (monocytes and neutrophils) as well as a decreased leukocyte infiltration. ...
Biodegradability and mechanical properties of magnesium alloys are attractive for orthopaedic and cardiovascular applications. In order to study their cytotoxicity usually bone cells are used. However, after implantation, diverse and versatile cells are recruited and interact. Among the first ones coming into play are cells of the immune system, which are responsible for the inflammatory reaction. Macrophages play a central role in the inflammatory process due to the production of cytokines involved in the tissue healing but also in the possible failure of the implants. In order to evaluate the in vitro influence of the degradation products of magnesium-based alloys on cytokine release, the extracts of pure magnesium and two magnesium alloys (with gadolinium and silver as alloying elements) were examined in an inflammatory in vitro model. Human promonocytic cells (U937 cells) were differentiated into macrophages and further cultured with magnesium-based extracts for 1 and 3 days (simulating early and late inflammatory reaction phases), either at 37°C or at 39°C (mimicking normal and inflammatory conditions, respectively). All extracts exhibit very good cytocompatibility on differentiated macrophages. Results suggest that M1 and even more M2 profiles of macrophage were stimulated by the extracts of Mg. Furthermore, Mg-10Gd and Mg-2Ag extracts introduced a nuancing effect by rather inhibiting macrophage M1 profile. Magnesium-based biomaterials could thus induce a faster inflammation resolution while improving tissue repair. Statement of Significance Macrophage are the key-cells during inflammation and can influence the fate of tissue healing and implant performance. Magnesium-based implants are biodegradable and bioactive. Here we selected an in vitro system to model early and late inflammation and effect of pyrexia (37°C versus 39°C). We showed the beneficial and nuancing effects of magnesium (Mg) and the selected alloying elements (silver (Ag) and gadolinium (Gd)) on the macrophage polarisation. Mg extracts exacerbated simultaneously the macrophage M1 and M2 profiles while Mg-2Ag and Mg-10Gd rather inhibited the M1 differentiation. Furthermore, 39°C exhibited protective effect by either decreasing cytokine production or promoting anti-inflammatory ones, with or without extracts. Mg-based biomaterials could thus induce a faster inflammation resolution while improving tissue repair.
... α7nAChR is commonly acknowledged to be widely expressed in immune cells such as macrophages as well as neuronal cells, thus playing a vital role in various kinds of diseases [4,5]. α7nAChR has been demonstrated to produce an alleviative effect in the suppression of inflammatory reaction, thus regulating the pathogenesis and progression of several inflammation and immune-related diseases, including atherosclerosis, myocardial infarction (MI), hypertension, inflammatory bowel disease, and multiple sclerosis [6][7][8][9]. In MI, although evidence has been provided to prove the therapeutic effect of activating α7nAChR in the treatment of myocardial infarction [10][11][12], the effect of α7nAChR in MI, also well known as heart attack, is still controversial and the specific mechanism is not yet clarified. ...
Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been previously reported to play an alleviative role in myocardial infarction (MI). In this study, we investigated its specific mechanism. α7nAChR−/− mice and its control (α7nAChR+/+) were used for the study of α7nAChR. Left anterior descending coronary artery occlusion was conducted for the creation of mice MI model and lipopolysaccharide (LPS) was used as inflammatory stressor in murine peritoneal macrophages. Triphenyltetrazolium chloride (TTC) staining and echocardiography was used for the detection of infarct size and cardiac function, respectively. Western blot was conducted for the testing of autophagy-related proteins and enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) was used for the testing of proinflammatory cytokines. Rapamycin was used for the induction of autophagy through inhibiting mammalian target of rapamycin (mTOR)-related signaling. We found that knocking out α7nAChR enhanced the cardiac infarct size and damaged cardiac function in MI. α7nAChR deficiency increased the levels of several proinflammatory cytokines in serum and spleen from MI mice as well as murine macrophages under inflammatory stress. α7nAChR deletion decreased the level of autophagy in spleen from MI mice and macrophages under inflammatory stress. Rapamycin alleviated the cardiac function and systemic inflammatory reaction in MI mice as well as inflammatory reaction in macrophages under inflammatory stress, which was attenuated by knocking out α7nAChR. Our current study investigated the mechanism of α7nAChR-mediated cardio-protective and anti-inflammatory effect related to mTOR-related autophagy, which might provide a novel insight in the treatment of MI.
... Recent studies found that the level of inflammatory cytokines is related to the damage of cardiac function and necrosis of cells post ischemic. The inflammatory response plays an important role in myocardial I/R injury, and it has been proved that the I/R-induced injury improved when inflammation was inhibited by the anti-inflammatory agents [7][8][9] . NF-κB (Nuclear factor-kappa B) is a nuclear protein factor, originally found in B-lymphocyte nuclear extracts, that binds to the immunoglobulin κ-chain gene enhancer κB sequence and generally presents in the cytoplasm in the form of a heterodimer (P50 and P65) [10] . ...
Myocardial ischemia-reperfusion (I/R) injury is a major contributor to the morbidity and mortality associated with coronary artery disease. How to ensure the recovery of blood supply to ischemic myocardial tissue while avoiding or reducing I/R injury remains a critical problem in clinical practice. In the present study, we examined the function of phospholipase C epsilon-1 (PLCE1) by an H9c2 H/R (H/R) model and a rat myocardial I/R injury model. The expression of PLCE1 and its effect on I/R injury-induced inflammatory response as well as its possible underlying mechanism were investigated. Our results have shown that PLCE1 was progressively increased along with the increasing of hypoxia time in the H/R H9c2 and HL-1 cells. In myocardial I/R rats, PLCE1 presented a low expression level in the sham group, however, it was increased sharply in the I/R group. Over-expression of PLCE1 promoted the expression of IL-6, TNF-α, and IL-1α, and decreased the expression of IL-10. Knockdown of PLCE1 decreased the expression of IL-6, TNF-α, and IL-1α, and increased the expression of IL-10. Furthermore, over-expression of PLCE1 increased the phosphorylation of p38, ERK1/2, and NF-κB P65 while knockdown of PLCE1 inhibited their phosphorylation. In conclusion, this study provided evidence that PLCE1 was up-regulated in H/R H9c2 cell and I/R rat. Over-expression of PLCE1 promoted the inflammatory via activation of the NF-κB signaling pathway.
... Gd-chelated derivatives are widely used as contrast agents for medical magnetic resonance imaging (Adding et al. 2006). Gd salts such as the trivalent cation gadolinium chloride (GdCl 3 ) bring about a wide variety of changes in Kupffer cell-related pathophysiological processes as observed in drug-induced liver toxicity, lung inflammation, and ischemia-reperfusion (Frid et al. 2006;Sindram et al. 2001;Singh et al. 2004;Strande et al. 2009). ...
... Consequently, increasing GdCl 3 concentrations leads to the sequential control of major neutrophil microbicidal functions. Thus, agreeing with previous data (Strande et al. 2009). ...
This study investigated the potential of gadolinium chloride (GdCl3), an inhibitor of kupffer cells on the myeloperoxidase (MPO) function, both in vivo on colon inflammation model and in vitro on thioglycollate-elicited peritoneal neutrophils. Colon inflammation was induced in mice (n = 7) by 4% acetic acid (AA) enema. GdCl3 (10 mg/kg) treatment was given 24 h before AA challenge. Clinical changes during the protocol were scored. Colons were segmented into distal and proximal parts for histological and biochemical assessment. Furthermore, myeloperoxidase (MPO) enzymes were extracted and analyzed by western blot. Short-term GdCl3 treatment inhibited dose-dependently superoxide anion (O2·−), alkaline phosphatase (ALP), and MPO release and promoted neutrophil apoptosis. In vivo, low-dose GdCl3 improved colitis scores and inhibited acute phagocyte recruitment and colon damage within the mucosa as revealed by the decrease in MPO, nitric oxide (NO), and malondialdehyde (MDA) levels. At the same time, GdCl3 restored catalase (CAT), superoxide dismutase (SOD) activities, and reduced glutathione (GSH) levels, thus reversing the MDA/GSH ratio in both distal and proximal colons. Compared to proximal, distal colon was more altered and displayed higher pathological manifestations. Lastly, the induction of apoptosis and regulation of the major nitrosative and oxidative functions of neutrophils by GdCl3 suggests its consideration as a beneficial tool in attenuating colon inflammation.
... Согласно представляемой гипотезе привлекательной идеей является возможность применения управляемой фармакологической блокады иммунных функций клеток Купфера для предупреждения развития неуправляемых реакций СВО. В экспериментальных работах для оценки влияния фагоцитоза на развитие и течение острого поражения легких и изучения возможности снижения воспалительных реакций, вызванных ишемией-реперфузией после резекций печени у крыс, блокатор функции печеночных макрофагов хлорид гадолиния в серии исследований продемонстрировал ослабление воспалительных реакций как in vitro, так и in vivo; при этом наблюдали увеличение секреции клетками Купфера противовоспалительных цитокинов [50]. В других экспериментальных работах было установлено снижение активации клеток Купфера при помощи гадолиния, блокаторов кальциевых каналов [51][52][53], пентоксифиллина [54] с целью повышения выживаемости трансплантированного органа. ...
Despite the fact that the key role of the liver in the formation of the immune response to injury is not in doubt, the mechanisms of weakening the immune response to infectious and noninfectious lesions in patients with hepatic failure remain unclear. We propose an original hypothesis of forming the ways to limit the amplitude of the systemic inflammatory response in patients with the end-stage liver disease. The basis of the hypothesis is the idea that as a result of reducing the intensity of the natural stimulation of membrane mCD14 receptors by the ligands of infectious nature, the basic mechanism of the systemic immune response induction by liver macrophages (Kupffer cells) is interrupted. According to the proposed hypothesis, in condition of liver failure, the synthesis of lipopolysaccharide-binding protein by hepatocytes is reduced. This leads to a decreased amplitude and intensity of the protective immune responses. This fact explains a number of clinical phenomena observed in patients with liver failure/dysfunction that consist in a reduced reactivity of the organism to the damage inflicted by infectious and noninfectious agents. The authors consider it possible to use this hypothesis in the search for new trends to prevent the immune system hyper-reactivity in sepsis, and to improve the therapeutic strategies for the management of patients at high risk of infectious complications after liver transplantation.Keywords: sepsis, systemic inflammatory response, Kupffer cells, hepatic failure, lipopolysaccharide, lipopolysac-charide-binding protein.
Paradox: Does liver insuffi ciency protect the patient? A hypothesis. Available from: https://www.researchgate.net/publication/315633798_Paradox_Does_liver_insuffi_ciency_protect_the_patient_A_hypothesis [accessed Jun 30, 2017].
... These results indicated a critical role of M1 microglia/macrophages in inducing the necroptosis of astrocytes after SCI. Considering that GdCl 3 also affects neutrophils [38], which are abundant in the injury epicenter after SCI, and that iNOS can also be expressed by neutrophils [39]. The beneficial effects of GdCl 3 treatment may also be contributed by the inhibition of neutrophils. ...
A unique feature of the pathological change after spinal cord injury (SCI) is the progressive enlargement of lesion area, which usually results in cavity formation and is accompanied by reactive astrogliosis and chronic inflammation. Reactive astrocytes line the spinal cavity, walling off the lesion core from the normal spinal tissue, and are thought to play multiple important roles in SCI. The contribution of cell death, particularly the apoptosis of neurons and oligodendrocytes during the process of cavitation has been extensively studied. However, how reactive astrocytes are eliminated following SCI remains largely unclear.
By immunohistochemistry, in vivo propidium iodide (PI)-labeling and electron microscopic examination, here we reported that in mice, reactive astrocytes died by receptor-interacting protein 3 and mixed lineage kinase domain-like protein (RIP3/MLKL) mediated necroptosis, rather than apoptosis or autophagy. Inhibiting receptor-interacting protein 1 (RIP1) or depleting RIP3 not only significantly attenuated astrocyte death but also rescued the neurotrophic function of astrocytes. The astrocytic expression of necroptotic markers followed the polarization of M1 microglia/macrophages after SCI. Depleting M1 microglia/macrophages or transplantation of M1 macrophages could significantly reduce or increase the necroptosis of astrocytes. Further, the inflammatory responsive genes Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) are induced in necroptotic astrocytes. In vitro antagonizing MyD88 in astrocytes could significantly alleviate the M1 microglia/macrophages-induced cell death. Finally, our data showed that in human, necroptotic markers and TLR4/MyD88 were co-expressed in astrocytes of injured, but not normal spinal cord.
Taken together, these results reveal that after SCI, reactive astrocytes undergo M1 microglia/macrophages-induced necroptosis, partially through TLR/MyD88 signaling, and suggest that inhibiting astrocytic necroptosis may be beneficial for preventing secondary SCI.
... GdCl 3 (10 mg/kg) has been demonstrated to exert a protective potential in I/R-induced brain injury and hepatic injury and to protect the myocardium against I/R-induced inflammation via the reduction of circulating monocytes and neutrophils and the infiltration of leukocytes. This dose also attenuated myocardial stunning when administered prior to the onset of ischemia or during ischemia, but it did not enhance the contractile function of normal myocardium [21][22][23][24] . However, the precise mechanism(s) underlying the effect of GdCl 3 are not known. ...
... In this study, we found that lowdose GdCl 3 dramatically inhibited AA-induced Ca 2+ overload and elevated resting [Ca 2+ ] i in A/R NRVMs ( Figure 3B-E). We also demonstrated that low-dose GdCl 3 significantly ameliorates I/R-induced cytochrome c release in vivo ( Figure 5H) and in vitro ( Figure 3A) and inhibits cell apoptosis via regulation of Previous studies have demonstrated that pathophysiological responses triggered after reperfusion include the release of activation factors and free radicals, which activate phospholipase A 2 and increase AA release [24] . The accumulated AA in the myocardium may play an important role in post-I/R injury because a time-dependent degradation of membrane phospholipids associated with an increase in membrane permeability was observed in the ischemic myocardium [17,38] . ...
Aim:
We have shown that low-dose gadolinium chloride (GdCl3) abolishes arachidonic acid (AA)-induced increase of cytoplasmic Ca(2+), which is known to play a crucial role in myocardial ischemia/reperfusion (I/R) injury. The present study sought to determine whether low-dose GdCl3 pretreatment protected rat myocardium against I/R injury in vitro and in vivo.
Methods:
Cultured neonatal rat ventricular myocytes (NRVMs) were treated with GdCl3 or nifedipine, followed by exposure to anoxia/reoxygenation (A/R). Cell apoptosis was detected; the levels of related signaling molecules were assessed. SD rats were intravenously injected with GdCl3 or nifedipine. Thirty min after the administration the rats were subjected to LAD coronary artery ligation followed by reperfusion. Infarction size, the release of serum myocardial injury markers and AA were measured; cell apoptosis and related molecules were assessed.
Results:
In A/R-treated NRVMs, pretreatment with GdCl3 (2.5, 5, 10 μmol/L) dose-dependently inhibited caspase-3 activation, death receptor-related molecules DR5/Fas/FADD/caspase-8 expression, cytochrome c release, AA release and sustained cytoplasmic Ca(2+) increases induced by exogenous AA. In I/R-treated rats, pre-administration of GdCl3 (10 mg/kg) significantly reduced the infarct size, and the serum levels of CK-MB, cardiac troponin-I, LDH and AA. Pre-administration of GdCl3 also significantly decreased the number of apoptotic cells, caspase-3 activity, death receptor-related molecules (DR5/Fas/FADD) expression and cytochrome c release in heart tissues. The positive control drug nifedipine produced comparable cardioprotective effects in vitro and in vivo.
Conclusion:
Pretreatment with low-dose GdCl3 significantly attenuates I/R-induced myocardial apoptosis in rats by suppressing activation of both death receptor and mitochondria-mediated pathways.
... Studies proved that TNF-α released first at the initial stage of ischemia reperfusion and its content was related to the severity as well as the prognosis of the disease [18]. The past studies on animals showed that it could reduce the number of circulating neutrophils and monocytes as well as their filtration to the damaged myocardial muscles and then to induce the clear protection action on the myocardial muscles through lifting the levels of TNF-α expression in the ischemic area prior to I/R [19]. Some other studies found the myocardial I/R led to the obvious increase of the IL-6 level in serum [20]. ...
Background:
Acute myocardial infarction (AMI) was a type of disease with high mortality rate and high disability rate. And about 50% of the final area of myocardial infarction after AMI was led by ischemia/reperfusion (I/R) injury. The I/R injury was a kind of systemic inflammatory response, in which the main performance laid in the release of the large quantity of inflammatory cytokines. The basic experiments, clinical studies and the large scaled epidemiology investigations found that the low functions of vagus nerves had close relevance with the occurrence, development and prognosis of the cardiovascular diseases. This study investigate the effects of cholinergic anti-inflammatory pathway with with vagus never stimulation I/R injury in canine.
Methods:
18 adult mongrel dogs were randomly divided into 3 groups (n = 6): sham operation group (sham Group), ischemia/reperfusion group (I/R group), right vagus nerve stimulation and ischemia/reperfusion group (STM group). The hemodynamic indexes were measured after reperfusion 120 min. Through internal jugular venous blood, serum acetylcholine (Ach), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) concentrations were detected by ELISA. Alpha 7 subunit Ach acetylcholine receptor (α7nAchR) expression level was detected with immunohistochemical method. HE staining was used to observe the degree of neutrophil infiltration.
Results:
After ischemia/reperfusion 120 min, compared with sham group, TNF-α and IL-6 were significantly decreased, Ach content increased, the expression of α7nAchR protein was significantly reduced in I/R group (P < 0.05). Expression of α7nAchR protein, Ach content, TNF-α and IL-6 level had no significant difference in STM group (P < 0.05). Compared with I/R group, the expression of Ach and α7nAchR protein significantly increased the TNF- and IL-6 levels decreased in STM group (P < 0.05). Compared with the baseline, TNF-α and IL-6 levels significantly increased Ach content decreased in I/R group after ischemia /reperfusion 120 min (P < 0.05). Ach, TNF-α and IL-6 levels had no significant change in sham group and STM group of (P < 0.05). TNF-α and IL-6 were negatively correlated with Ach in I/R group (P < 0.05), and TNF-α, IL-6 were negatively correlated with Ach in group STM (P < 0.05). Massive infiltration of neutrophils were detected in myocardial tissue of I/R group, and a small number of neutrophils infiltration were detected in STM group.
Conclusion:
Right vagus nerve stimulation could activate anti-inflammatory pathway and inhibit the systemic and local inflammatory reaction to relieve myocardial I/R injury.
